Numerous bioproducts including primary and secondary metabolites, recombinant proteins, and other biopolymers are produced by microbial fermentation. However, natural organisms are not optimized for this task, and therefore microorganisms are often engineered to enhance their metabolic capacities.
The cofactor pair NADPH/NADP+ is essential for all living organisms. Their importance centers mainly on their use as donors and/or acceptors of reducing equivalents in many oxidation-reduction reactions in living cells. The NADH/NAD+ pair is used for catabolic activities of the cell, whereas anabolic metabolism specifically requires NADPH/NADP+. Together, these nucleotides have a direct impact on virtually every oxidation-reduction metabolic pathway in the cell.
Many industrially useful compounds require NADPH for their synthesis. Due to the high cost and regeneration difficulty of these coenzymes, the enzymatic production of cofactor-dependent compounds has proved to be challenging.
Polyhydroxyalkanoates (PHAs) are a family of biodegradable polyesters synthesized by numerous microorganisms and function as an intracellular carbon and energy storage material. The best characterized member of PHA is poly(3-hydroxybutyric acid), PHB. PHB is synthesized from acetyl-CoA in three sequential reaction steps catalyzed by the enzymes of the phb operon, β-ketothiolase, acetoacetyl-CoA reductase and PHB synthase. The second reaction catalyzed by the reductase requires NADPH as a cofactor. In other words, the requirement for NADPH plays a key role in the biosynthesis of important metabolites such as PHB.
NADH/NADP+ transhydrogenase, the enzyme responsible for the conversion of NADH to NADPH, can be induced in E. coli strains following exposure to agents, such as H2O2. This represents one method of increasing the intracellular levels of NADPH. However, such methods are not useful due to the deleterious effects of the agents. Therefore, methods of increasing the intracellular levels of the cofactor NADPH is required that do not otherwise damage the cell or detract from its ability to produce large amounts of desirable protein.